Project Summary/Abstract The goal of my laboratory is to define the molecular mechanisms by which accurate cell division occurs. Our efforts focus on the kinetochore, the central player in directing chromosome segregation. The kinetochore is a macromolecular structure that connects chromosomes to the microtubule polymers that power their movement. Our goal is to generate a coherent model for how the kinetochore functions as an integrated molecular machine. To direct faithful chromosome segregation, kinetochores must form two key interaction interfaces. First, kinetochores must associate with a single site on each chromosome to direct the assembly of a stable kinetochore structure. In vertebrates, this site is defined epigenetically by the presence of a specialized histone variant termed CENP-A, and through contributions of a 16-subunit Constitutive Centromere-Associated Network (CCAN). Together, these proteins form the interface with centromeric chromatin. Despite the identification of these molecules, it remains unclear how the CCAN is established and reorganized during the cell cycle, and also how these processes are modulated during different cell division programs, such as in the context of meiosis and early development. In addition, centromeres must have a specific open chromatin environment to facilitate proper kinetochore function, but the relationship between the CCAN and centromere chromatin is poorly defined. Second, kinetochores must form robust interactions with dynamic microtubule polymers and harness the force generated by depolymerizing microtubules to direct chromosome segregation. To understand this elegant interface, it is critical to define the individual contributions of key outer kinetochore microtubule-binding complexes and also assess their integrated activities. The kinetochore must also sense and correct microtubule attachments to ensure high fidelity chromosome segregation, requiring the functions from the spindle assembly checkpoint components. To understand these critical kinetochore activities and the functional requirements for chromosome segregation, it is also important to define the complete complement of human genes that are required for chromosome segregation. The advent of CRISPR/Cas9-based genome editing has transformed the capability to conduct functional genetics experiments in human cells. This includes the ability to systematically screen gene targets for their loss of function phenotypes using cell biological assays and genome-wide functional genetics screening to analyze context-dependent essentiality to define synthetic lethality relationships. For the work in this proposal, our lab will investigate the fundamental mechanisms of chromosome segregation and kinetochore function, focusing on three related areas: 1) Specification and formation of the centromere- DNA interface, 2) Generation and regulation of dynamic kinetochore-microtubule interactions, 3) Functional genetic approaches to analyze chromosome segregation. We will analyze key open questions in these important areas using combined cell biological, biochemical, proteomic, and functional genetics approaches.